U.S. patent application number 15/118113 was filed with the patent office on 2017-06-15 for treatment of h. pylori infections using mtan inhibitors.
This patent application is currently assigned to ALBERT EINSTEIN COLLEGE OF MEDICINE, INC.. The applicant listed for this patent is ALBERT EINSTEIN COLLEGE OF MEDICINE, INC., VICTORIA LINK LIMITED. Invention is credited to Keith Clinch, Shivali Ashwin Gulab, Vern L. Schramm.
Application Number | 20170166571 15/118113 |
Document ID | / |
Family ID | 53800544 |
Filed Date | 2017-06-15 |
United States Patent
Application |
20170166571 |
Kind Code |
A1 |
Schramm; Vern L. ; et
al. |
June 15, 2017 |
TREATMENT OF H. PYLORI INFECTIONS USING MTAN INHIBITORS
Abstract
Methods of treating infections due to Helicobacter pylori (H.
pylori), in particular in subjects having a peptic ulcer, are
disclosed where the methods comprise administering inhibitors of H.
pylori MTAN (5'-methylthioadenosine nucleosidase) to the
subject.
Inventors: |
Schramm; Vern L.; (New
Rochelle, NY) ; Clinch; Keith; (Lower Hutt, NZ)
; Gulab; Shivali Ashwin; (Wellington, NZ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALBERT EINSTEIN COLLEGE OF MEDICINE, INC.
VICTORIA LINK LIMITED |
Bronx
Wellington |
NY |
US
NZ |
|
|
Assignee: |
ALBERT EINSTEIN COLLEGE OF
MEDICINE, INC.
Bronx
NY
|
Family ID: |
53800544 |
Appl. No.: |
15/118113 |
Filed: |
February 6, 2015 |
PCT Filed: |
February 6, 2015 |
PCT NO: |
PCT/US15/14778 |
371 Date: |
August 11, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61938755 |
Feb 12, 2014 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
Y02A 50/30 20180101;
C07D 487/04 20130101; A61P 31/04 20180101; C07D 471/04 20130101;
A61K 31/519 20130101 |
International
Class: |
C07D 487/04 20060101
C07D487/04 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
number GM41916 awarded by the National Institutes of Health and
under Center for Synchrotron Biosciences grant number P30-EB-009998
awarded by the National Institute of Biomedical Imaging and
Bioengineering (NIBIB). The government has certain rights in the
invention.
Claims
1. A method of treating a Helicobacter pylori (H. pylori) infection
in a subject comprising administering to the subject a compound of
formula (I) in an amount effective to inhibit growth of H. pylori,
wherein formula (I) is ##STR00066## wherein R is Q or CH.sub.2SQ,
wherein Q is C1-C9 alkyl, aryl, heteroaryl, aralkyl, or C4-C7
cycloalkyl, and wherein Q is optionally substituted with one or
more halogen, OH and/or NH.sub.2 groups, or a pharmaceutically
acceptable salt thereof, or an ester thereof.
2. The method of claim 1, wherein the compound has the formula
##STR00067##
3. The method of claim 1, wherein R is Q.
4. The method of claim 1, wherein R is CH.sub.2SQ.
5. The method of claim 1, wherein Q is C1-C9 alkyl.
6. The method of claim 1, wherein Q is C4-C7 cycloalkyl.
7. The method of claim 1, wherein Q is aryl.
8. The method of claim 1, wherein Q is heteroaryl.
9. The method of claim 1, wherein Q is aralkyl.
10. The method of claim 1, wherein Q is aryl, heteroaryl or aralkyl
substituted with a halogen.
11. The method of claim 1, wherein R is Q or CH.sub.2SQ, wherein Q
is C2-C9 linear alkyl, aryl, heteroaryl, aralkyl, or C4-C7
cycloalkyl, and wherein Q is optionally substituted with one or
more halogen, OH and/or NH.sub.2 groups.
12. The method of claim 10, wherein the halogen is at an ortho,
meta or para position.
13. The method of claim 1, wherein the halogen is Cl, F, Br or
I.
14. The method of claim 1, wherein Q is C3-C9 linear alkyl or
heteroaryl.
15. The method of claim 1, wherein the compound is selected from
the group consisting of ##STR00068## ##STR00069## or a
pharmaceutically acceptable salt thereof, or an ester thereof.
16. A method of treating a Helicobacter pylori (H. pylori)
infection in a subject comprising administering to the subject a
compound in an amount effective to inhibit growth of H. pylori,
wherein the compound is selected from the group consisting of
##STR00070## ##STR00071## ##STR00072## or a pharmaceutically
acceptable salt thereof, or an ester thereof.
17. The method of claim 1, wherein the compound is administered in
an amount that is effective to inhibit H. pylori
5'-methylthioadenosine nucleosidase (MTAN).
18. The method of claim 1, wherein the compound inhibits growth of
H. pylori but does not inhibit the growth of one or more bacterium
selected from the group consisting of E. coli, V. cholerae, S.
aureus, K. pneumoniae, S. flexneri, S. enterica and P.
aeruginosa.
19. The method of claim 18, wherein the compound does not inhibit
the growth of all of E. coli, V. cholerae, S. aureus, K.
pneumoniae, S. flexneri, S. enterica and P. aeruginosa.
20. The method of claim 1, wherein the compound is more effective
in inhibiting growth of H. pylori than amoxicillin, metronidazole
or tetracyclin.
21. The method of claim 1, wherein the subject has a peptic
ulcer.
22. The method of claim 1, wherein the subject has a gastric ulcer
or a duodenal ulcer.
23. The method of claim 1, wherein the compound is administered
orally.
24. A compound having the structure ##STR00073## ##STR00074##
##STR00075## or a pharmaceutically acceptable salt thereof, or an
ester thereof.
25. A pharmaceutical composition comprising a compound of claim 24
and a pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional
patent application No. 61/938,755, filed on Feb. 12, 2014, the
contents of which is herein incorporated by reference in its
entirety.
FIELD OF THE INVENTION
[0003] The invention relates to treating infections due to
Helicobacter pylori (H. pylori) using inhibitors of H. pylori MTAN
(5'-methylthioadenosine nucleosidase), in particular in subjects
having a peptic ulcer.
BACKGROUND OF THE INVENTION
[0004] Throughout this application various publications are
referred to in superscripts. Full citations for these references
may be found at the end of the specification before the claims. The
disclosures of these publications are hereby incorporated by
reference in their entireties into the subject application to more
fully describe the art to which the subject application
pertains.
[0005] H. pylori is a gram-negative bacterium and lives
microaerophilically in the gastric mucosa of its human host. It is
related to 85 percent of gastric and 95 percent of duodenal
ulcers.sup.1. Drug resistance is prevalent in clinical isolates of
H. pylori. After less than thirty years of specific antibiotic
treatment, it is increasingly difficult to eradicate H. pylori
using a combination of two antibiotics with two weeks
therapy.sup.2. Antibiotics with new targets and mechanisms of
action are needed to treat H. pylori infections.
[0006] Gram negative bacteria are dependent on menaquinone as
electron transporters in respiration and have maintained
biosynthetic pathways for these essential metabolites.sup.3. In
contrast, humans lack the pathway of menaquinone synthesis, and
targeting the menaquinone pathway provides an anti-bacterial drug
design approach. Recently, a menaquinone synthetic pathway has been
proposed in Campylobacter and Helicobacter that differs from most
bacteria.sup.4'5. In this pathway, 6-amino-6-deoxyfutalosine is
synthesized by MqnA and cleaved at the N-ribosidic bond by a MTAN
with specificity also extending to 5'-methylthioadenosine and
adenosylhomocysteine as well as 6-amino-6-deoxyfutalosine. HpMTAN
converts 6-amino-6-deoxyfutalosine to adenine and dehypoxanthine
futalosine, the latter being used as the processor of menaquinone
synthesis. The early reactions of this pathway do not exist in the
normal bacterial flora of humans, making enzymes catalyzing these
reactions appealing drug targets. HpMTAN is closely related to the
5'-methylthioadenosine/S-adenosylhomocysteine hydrolases (MTANs)
found in other bacteria. The well-characterized MTANs are
associated with quorum sensing and S-adenosylmethionine recycling
in most species and are not essential for bacterial growth.sup.6.
Transition state analogue inhibitors of picomolar to femtomolar
affinity have been developed to interrupt bacterial functions
associated with quorum sensing.sup.6,7
[0007] The present invention addresses the need for new compounds
that selectively block the growth of H. pylori.
SUMMARY OF THE INVENTION
[0008] The invention provides methods of treating a Helicobacter
pylori (H. pylori) infection in a subject comprising administering
to the subject a compound of formula (I) in an amount effective to
inhibit growth of H. pylori, wherein formula (I) is
##STR00001##
[0009] wherein R is Q or CH.sub.2SQ, wherein Q is C1-C9 alkyl aryl,
heteroaryl, aralkyl, or C4-C7 cycloalkyl and wherein Q is
optionally substituted with one or more halogen, OH and/or NH.sub.2
groups,
[0010] or a pharmaceutically acceptable salt thereof, or an ester
thereof.
[0011] The invention further provides a compound having the
structure:
##STR00002## ##STR00003## ##STR00004##
[0012] or a pharmaceutically acceptable salt thereof, or an ester
thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1. Concentration of Hexyl-SerMe-Immucillin A in mouse
gastric mucin versus time following a single dose of 10 mg/kg
PO.
DETAILED DESCRIPTION OF THE INVENTION
[0014] The invention provides a method of treating a Helicobacter
pylori (H. pylori) infection in a subject comprising administering
to the subject a compound of formula (I) in an amount effective to
inhibit growth of H. pylori, wherein formula (I) is
##STR00005##
[0015] wherein R is Q or CH.sub.2SQ, wherein Q is C1-C9 alkyl,
aryl, heteroaryl, aralkyl, or C4-C7 cycloalkyl, and wherein Q is
optionally substituted with one or more halogen, OH and/or NH.sub.2
groups,
[0016] or a pharmaceutically acceptable salt thereof, or an ester
thereof.
[0017] Preferred compounds include those having the formula
##STR00006##
[0018] Q can be, for example, C1-C9 alkyl, e.g., C1-C5 alkyl; e.g.,
a methyl (Me), ethyl (Et), propyl (Pr), butyl or pentyl group. Q
can be, for example, C4-C7 cycloalkyl, i.e., C4 cycloalkyl, C5
cycloalkyl, C6 cycloalkyl, or C7 cycloalkyl. Q can be, for example,
aryl. The term "aryl" means an aromatic radical having 4 to 12
carbon atoms. Examples include phenyl, 1-naphthyl and 2-naphthyl.
"Heteroaryl" means a 4 to 12 member ring that includes one or more
N, S, or O atoms in the ring. Examples include imidazol-4-yl,
imidazol-2-yl, thiazol-2-yl, thiazol-4-yl, thiazol-5-yl,
pyridin-2-yl, pyridine-3-yl, pyridine-4-yl and pyrazin-2-yl.
Preferred aryls and heteroaryls include those having 5 or 6 members
in the ring. Preferably, the aralkyl includes a C1-C3 alkyl group
and a 4-6 membered ring, which can include heteroatoms.
[0019] Q can be substituted with one or more halogen, hydroxyl or
NH.sub.2 groups. Preferred halogens are Cl, F, Br or I. Chlorine
and fluorine are more preferred halogens. The substitution can be
at an ortho, meta or para position.
[0020] Preferred compounds include those where R is Q or
CH.sub.2SQ, wherein Q is C2-C9 linear alkyl, aryl, heteroaryl,
aralkyl, or C4-C7 cycloalkyl, and wherein Q is optionally
substituted with one or more halogen, OH and/or NH.sub.2 groups.
Preferred compounds also include those where Q is C3-C9 linear
alkyl or heteroaryl.
[0021] Preferred compounds include those selected from the group
consisting of
##STR00007## ##STR00008## ##STR00009##
[0022] or a pharmaceutically acceptable salt thereof, or an ester
thereof.
[0023] According, the invention provides a method of treating a
Helicobacter pylori (H. pylori) infection in a subject comprising
administering to the subject a compound in an amount effective to
inhibit growth of H. pylori, wherein the compound is selected from
the group consisting of:
##STR00010## ##STR00011## ##STR00012##
[0024] or a pharmaceutically acceptable salt thereof, or an ester
thereof.
[0025] The invention further provides a compound having the
structure:
##STR00013## ##STR00014## ##STR00015##
or a pharmaceutically acceptable salt thereof, or an ester
thereof.
[0026] The term "pharmaceutically acceptable salts" includes
non-toxic salts derived from inorganic or organic acids, including,
for example, the following acid salts: acetate, adipate, alginate,
aspartate, benzoate, benzenesulfonate, bisulfate, butyrate,
citrate, camphorate, camphorsulfonate, cyclopentanepropionate,
digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,
glucoheptanoate, glycerophosphate, glycolate, hemisulfate,
heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide,
2-hydroxyethanesulfonate, lactate, maleate, malonate,
methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,
oxalate, palmoate, pectinate, persulfate, 3-phenylpropionate,
phosphate, picrate, pivalate, propionate, p-toluenesulfonate,
salicylate, succinate, sulfate, tartrate, thiocyanate, and
undecanoate.
[0027] Preferably, the compound is administered in an amount that
is effective to inhibit H. pylori 5'-methylthioadenosine
nucleosidase (MTAN).
[0028] Preferably, the compound inhibits growth of H. pylori but
does not inhibit the growth of one or more bacterium selected from
the group consisting of E. coli, V. cholerae, S. aureus, K.
pneumoniae, S. flexneri, S. enterica and P. aeruginosa. More
preferably, the compound does not inhibit the growth of all of E.
coli, V. cholerae, S. aureus, K. pneumoniae, S. flexneri, S.
enterica and P. aeruginosa. Preferably, the compound is more
effective in inhibiting growth of H. pylori than is amoxicillin,
metronidazole or tetracyclin.
[0029] Preferably, the subject has a peptic ulcer, such as a
gastric ulcer or a duodenal ulcer.
[0030] Preferably, the compound is administered orally. For oral
administration, the compound can be formulated into solid or liquid
preparations, for example tablets, capsules, powders, solutions,
suspensions and dispersions. The compound can be formulated with
agents such as, e.g., lactose, sucrose, corn starch, gelatin,
potato starch, alginic acid and/or magnesium stearate.
[0031] The compound can also be administered to a subject by other
routes known in the art, such as, e.g., parenterally, by
inhalation, topically, rectally, nasally, buccally or via an
implanted reservoir. The compound can be administered by means of
sustained release.
[0032] The invention further provides for the use a compound that
inhibits Helicobacter pylori (H. pylori) MTAN for the preparation
of a medicament for treating a H. pylori infection. The invention
still further provides a compound that inhibits Helicobacter pylori
(H. pylori) MTAN for use for treating a H. pylori infection.
[0033] The invention further provides for the use a compound that
inhibits Helicobacter pylori (H. pylori) MTAN for the preparation
of a medicament for treating a peptic ulcer. The invention still
further provides a compound that inhibits Helicobacter pylori (H.
pylori) MTAN for use for treating a peptic ulcer.
[0034] The present methods can also be applied to treating
infections due to other Helicobacter species and to Campylobacter
species, such as C. jejuni.
[0035] The invention further provides a pharmaceutical composition
comprising any of the compounds disclosed herein and a
pharmaceutically acceptable carrier. As used herein, a
"pharmaceutically acceptable carrier" is (i) compatible with the
other ingredients of the composition without rendering the
composition unsuitable for its intended purpose, and (ii) suitable
for use with subjects as provided herein without undue adverse side
effects (such as toxicity, irritation, and allergic response). Side
effects are "undue" when their risk outweighs the benefit provided
by the composition. Non-limiting examples of pharmaceutically
acceptable carriers include any of the standard pharmaceutical
carriers such as phosphate buffered saline solutions, water, and
emulsions such as oil/water emulsions and microemulsions.
[0036] This invention will be better understood from the
Experimental Details that follow. However, one skilled in the art
will readily appreciate that the specific methods and results
discussed are merely illustrative of the invention as described
more fully in the claims that follow thereafter.
EXPERIMENTAL DETAILS
Example 1
Materials and Methods
[0037] Materials. H. pylori (J99 and 43504), K. pneumoniae, S.
flexneri, S. enterica, S. aureus and P. aeruginosa were purchased
from the American Type Culture Collection. Defibrinated horse blood
(DHB) was from Hemostat Laboratories (Dixon, Calif.). Tryptic soy
agar (TSA) was purchased from Becton Dickinson and Company (Sparks,
Md.). MacConkey agar was from Oxoid LTD. (Basingstoke, Hampshire,
England). Xanthine oxidase and 5'-methylthioadenosine were
purchased from Sigma-Aldrich (St Louis, Mo.). The rest of the
materials were purchased with the highest purity available.
[0038] HpMTANpurification. The purification procedure of HpMTAN was
described previously.sup.10. Briefly, BL21 (DE3) cells harboring a
plasmid encoding HpMTAN with an N-terminal His6-tag were grown to
an optical density of 0.7 measured at 595 nm and IPTG was
introduced to a final concentration of 0.5 mM. After another 15 h
at 22.degree. C., cells were collected by centrifugation. The
pellet was suspended and later disrupted by pressure cell and
sonication. The soluble portion was applied to a Ni-NTA column and
HpMTAN was eluted with an imidazole concentration gradient of 200
to 500 mM. The protein was desalted using a Superdex G15
gel-filtration column then equilibrated and concentrated in 10 mM
Hepes, 30 mM KCl, pH 7.6. The purity was confirmed by SDS-PAGE.
[0039] K.sub.i determination. Kinetics of HpMTAN were determined
using a direct assay involving absorbance decrease at 274 nm
continuously as a consequence of formation of free adenine from
5'-methylthioadenosine. The K.sub.i and K.sub.i* values were
determined using coupled assays, in which xanthine oxidase was used
as the coupling enzyme and absorbance increase was followed at 292
nm as the product adenine is converted to 2,8-dihydroxyadenine.
Both assays have been previously described.sup.8.
[0040] Bacterial Growth.
[0041] H. pylori were grown for 72 hours under microaerophilic
condition (5% O.sub.2, 10% CO.sub.2 and 85% N.sub.2) at 37.degree.
C. on tryptic soy agar with 5% horse blood. To determine the MIC
values, the test substance was added to the gel solution right
before pouring. To compare the zones of inhibition, specific
antibiotics were added to the center of disc after spreading H.
pylori, and then H. pylori was allowed to grow for 72 hours under
microaerophilic condition at 37.degree. C.
[0042] General Experimental for Compounds.
[0043] All reactions were performed under an argon atmosphere.
Organic solutions were dried over anhydrous MgSO.sub.4 and the
solvents were evaporated under reduced pressure. Anhydrous and
chromatography solvents were obtained commercially and used without
any further purification. Potassium tert-butoxide was sublimed at
220.degree. C./0.1 torr. Thin layer chromatography (TLC) was
performed on glass or aluminum sheets coated with 60 F.sub.254
silica gel. Organic compounds were visualized under UV light and/or
a dip of 0.1% ninhydrin in EtOH, Ehrlich's solution or ammonium
molybdate (5 mass %) and cerium(IV) sulfate 4 H.sub.2O (0.2 mass %)
in aq. H.sub.2SO.sub.4 (2 M). Chromatography (flash column or an
automated system with continuous gradient facility) was performed
on silica gel (40-63 .mu.m). Optical rotations were recorded at a
path length of 1 dm and are in units of 10.sup.-1 deg cm.sup.2
g.sup.-1; concentrations are in g/100 mL. .sup.1H NMR spectra were
measured in CDCl.sub.3 or CD.sub.3OD (internal Me.sub.4Si, .delta.
0) and .sup.13C NMR spectra in CDCl.sub.3 (center line as stated)
or CD.sub.3OD (center line as stated). Assignments of .sup.1H and
.sup.13C resonances were based on 2D (.sup.1H-.sup.1H DQF-COSY,
.sup.1H-.sup.13C HSQC) and DEPT experiments. Abbreviations used: s,
singlet, d, doublet, t, triplet, q, quartet, bs, broad singlet, bt,
broad triplet, dd, doublet of doublets, ddd, doublet of doublets of
doublets, dt, doublet of triplets. High resolution electrospray
mass spectra (ESI-HRMS) were recorded on a Q-TOF Tandem Mass
Spectrometer.
Example A. Synthesis of
2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]ethan-1-ol
(A.1)
##STR00016##
[0044]
2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]ethan-1-o-
l (A.1)
[0045] 2-Aminoethanol (0.099 mL, 1.64 mmol), 9-deazaadenine (0.220
g, 1.64 mmol) and aq. formaldehyde solution (37%, 0.15 mL, 1.99
mmol) were stirred together in tert-butanol (3 mL) at 70.degree. C.
for 16 h. Silica gel was added to absorb all the solvent then the
solvent was evaporated and the residue purified by chromatography
on silica gel (CHCl.sub.3-MeOH-28% aq.NH.sub.4OH, 70:25:5).
Fractions containing product were evaporated and the residue
chromatographed again on silica gel (2-PrOH-28% aq. NH.sub.4OH,
92:8) to give A.1 as a colourless solid (0.101 g, 30%). .sup.1H NMR
(500 MHz, CD.sub.3OD): .delta. 8.16 (s, 1H), 7.47 (s, 1H), 3.95 (s,
2H), 3.68 (t, J=5.6 Hz, 2H), 2.78 (t, J=5.6 Hz, 2H). .sup.13C NMR
(125.7 MHz, CD.sub.3OD, centre line .delta. 49.0): .delta. 152.1
(C), 150.9 (CH), 146.6 (C), 129.0 (CH), 115.4 (C), 114.4 (C), 61.6
(CH.sub.2), 51.6 (CH.sub.2), 43.4 (CH.sub.2). ESI-HRMS calcd for
C.sub.9H.sub.14N.sub.5O.sup.+, (M+H).sup.+, 208.1193. found
208.1192.
Example B. Synthesis of
(2S)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]propan-1-o-
l (B.1)
##STR00017##
[0046]
(2S)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]prop-
an-1-ol (B.1)
[0047] (2S)-2-Aminopropan-1-ol (0.120 g, 1.60 mmol), 9-deazaadenine
(0.179 g, 1.33 mmol) and aq. formaldehyde solution (37%, 0.12 mL,
1.60 mmol) were stirred together in tert-butanol (3 mL) at
70.degree. C. for 16 h. Silica gel was added to absorb all the
solvent then the solvent was evaporated and the residue purified by
chromatography on silica gel (CHCl.sub.3-MeOH-28% aq.NH.sub.4OH,
80:18.5:1.5) to afford B.1 as a colourless solid (0.180 g, 61%).
.sup.1H NMR (500 MHz, CD.sub.3OD): .delta. 8.16 (s, 1H), 7.47 (s,
1H), 4.01 (d, J=13.6 Hz, 1H), 3.92 (d, J=13.6 Hz, 1H), 3.54 (dd,
J=11.0, 4.8 Hz, 1H), 3.43 (dd, J=11.0, 7.0 Hz, 1H), 2.84 (m, 1H),
1.09 (d, J=6.5 Hz, 3H). .sup.13C NMR (125.7 MHz, CD.sub.3OD, centre
line .delta. 49.0): .delta. 152.1 (C), 150.9 (CH), 146.5 (C), 128.9
(CH), 115.4 (C), 114.5 (C), 66.6 (CH.sub.2), 54.8 (CH), 41.0
(CH.sub.2), 16.5 (CH.sub.3). ESI-HRMS calcd for
C.sub.10H.sub.16N.sub.5O.sup.+, (M+H).sup.+, 222.1350. found,
222.1349.
Example C. Synthesis of
(2S)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]butan-1-ol
(C.1)
##STR00018##
[0048]
(2S)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]buta-
n-1-ol (C.1)
[0049] (2S)-2-Aminobutan-1-ol (0.100 g, 1.12 mmol), 9-deazaadenine
(0.150 g, 1.12 mmol) and aq. formaldehyde solution (37%, 0.101 mL,
1.35 mmol) were stirred together in tert-butanol (3 mL) at
70.degree. C. for 16 h. Silica gel was added to absorb all the
solvent then the solvent was evaporated and the residue purified by
chromatography on silica gel (CHCl.sub.3-7M NH.sub.3/MeOH, 9:1 then
85:15) to afford C.1 as a colourless solid (0.133 g, 50%). .sup.1H
NMR (500 MHz, CD.sub.3OD): .delta. 8.16 (s, 1H), 7.47 (s, 1H), 3.99
(d, J=13.6 Hz, 1H), 3.95 (d, J=13.6 Hz, 1H), 3.67 (dd, J=11.2, 4.4
Hz, 1H), 3.48 (dd, J=11.2, 6.5 Hz, 1H), 2.61 (m, 1H), 1.62-1.53 (m,
1H), 1.51-1.42 (m, 1H), 0.91 (t, J=7.5 Hz, 3H). .sup.13C NMR (125.7
MHz, CD.sub.3OD, centre line .delta. 49.0): .delta. 152.1 (C),
150.8 (CH), 146.6 (C), 128.9 (CH), 115.4 (C), 114.8 (C), 63.9
(CH.sub.2), 60.9 (CH), 41.1 (CH.sub.2), 24.6 (CH.sub.2), 10.7
(CH.sub.3). ESI-HRMS calcd for C.sub.11H.sub.17N.sub.5NaO.sup.+,
(M+Na).sup.+, 258.1326. found 258.1321.
Example D. Synthesis of
(2S)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]pentan-1-o-
l (D.1)
##STR00019##
[0050]
(2S)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]pent-
an-1-ol (D.1)
[0051] (2S)-2-Aminopentan-1-ol (0.050 g, 0.48 mmol), 9-deazaadenine
(0.065 g, 0.48 mmol) and aq. formaldehyde solution (37%, 0.044 mL,
0.59 mmol) were stirred in tert-butanol (2 mL) at 70.degree. C.
overnight. Silica gel was added to absorb all the solvent then the
solvent was evaporated and the residue purified by chromatography
on silica gel (CHCl.sub.3-7M NH.sub.3/MeOH, 9:1 then 85:15) to
afford D.1 as a colourless solid (0.073 g, 60%). .sup.1H NMR (500
MHz, CD.sub.3OD): .delta. 8.16 (s, 1H), 7.47 (s, 1H), 3.99 (d,
J=13.7 Hz, 1H), 3.96 (d, J=13.7 Hz, 1H), 3.66 (dd, J=11.3, 4.4 Hz,
1H), 3.48 (dd, J=11.3, 6.6 Hz, 1H), 2.69 (m, 1H), 1.54-1.30 (m,
4H), 0.89 (t, J=7.2 Hz, 3H). .sup.13C NMR (125.7 MHz, CD.sub.3OD,
centre line .delta. 49.0): .delta. 152.1 (C), 150.8 (CH), 146.6
(C), 128.9 (CH), 115.4 (C), 114.7 (C), 62.3 (CH.sub.2), 59.2 (CH),
41.1 (CH.sub.2), 34.3 (CH.sub.2), 20.3 (CH.sub.2), 14.6 (CH.sub.3).
ESI-HRMS calcd for C.sub.12H.sub.20N.sub.5O.sup.+, (M+H).sup.+,
250.1663. found 250.1663.
Example E. Synthesis of
(2S)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]hexan-1-ol
(E.1)
##STR00020##
[0052]
(2S)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]hexa-
n-1-ol (E.1)
[0053] (2S)-2-Aminohexan-1-ol (0.100 g, 0.85 mmol), 9-deazaadenine
(0.114 g, 0.85 mmol) and aq. formaldehyde solution (37%, 0.077 mL,
1.02 mmol) were stirred together at 70.degree. C. in tert-butanol
(3 mL) for 16 h. Silica gel was added to absorb all the solvent
then the solvent was evaporated and the residue purified by
chromatography on silica gel (CHCl.sub.3-7M NH.sub.3/MeOH, 9:1 then
85:15) to afford E.1 as a colourless solid (0.112 g, 50%). .sup.1H
NMR (500 MHz, CD.sub.3OD): .delta. 8.16 (s, 1H), 7.47 (s, 1H), 3.97
(s, 2H), 3.65 (dd, J=11.2, 4.4 Hz, 1H), 3.48 (dd, J=11.2, 6.6 Hz,
1H), 2.66 (m, 1H), 1.55-1.38 (m, 2H), 1.32-1.22 (m, 4H), 0.88 (t,
J=7.1 Hz, 3H). .sup.13C NMR (125.7 MHz, CD.sub.3OD, centre line
.delta. 49.0): .delta. 152.1 (C), 150.8 (CH), 146.6 (C), 128.9
(CH), 115.4 (C), 114.7 (C), 64.4 (CH.sub.2), 59.3 (CH), 41.2
(CH.sub.2), 31.7 (CH.sub.2), 29.3 (CH.sub.2), 23.9 (CH.sub.2), 14.3
(CH.sub.3). ESI-HRMS calcd for C.sub.13H.sub.21N.sub.5NaO.sup.+,
(M+Na).sup.+, 286.1644. found 286.1644.
Example F. Synthesis of
(2R)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]hexan-1-ol
(F.1)
##STR00021##
[0054]
(2R)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]hexa-
n-1-ol (F.1)
[0055] (2R)-2-Aminohexan-1-ol (0.100 g, 0.85 mmol), 9-deazaadenine
(0.114 g, 0.85 mmol) and aq. formaldehyde solution (37%, 0.077 mL,
1.02 mmol) were stirred together at 70.degree. C. in tert-butanol
(3 mL) for 16 h. Silica gel was added to absorb all the solvent
then the solvent was evaporated and the residue purified by
chromatography on silica gel (CHCl.sub.3-7M NH.sub.3/MeOH, 9:1 then
85:15) to afford F.1 as a colourless solid (0.108 g, 48%). The
.sup.1H and .sup.13C NMR were identical to the enantiomer E.1.
ESI-HRMS calcd for C.sub.13H.sub.22N.sub.5O.sup.+ (M+H).sup.+,
264.1819. found 264.1717.
Example G. Synthesis of
(2S)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-methylb-
utan-1-ol (G.1)
##STR00022##
[0056]
(2S)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-m-
ethylbutan-1-ol (G.1)
[0057] (2S)-2-Amino-3-methylbutan-1-ol (0.100 g, 0.97 mmol)
9-deazaadenine (0.130 g, 0.97 mmol), and aq. formaldehyde solution
(37%, 0.087 mL, 1.16 mmol) were stirred in tert-butanol (3 mL) at
70.degree. C. for 16 h. Silica gel was added to absorb all the
solvent then the solvent was evaporated and the residue purified by
chromatography on silica gel (CHCl.sub.3-7M NH.sub.3/MeOH, 93:7
then 85:15) to give G.1 as a colourless solid (0.082 g, 34%).
.sup.1H NMR (500 MHz, CD.sub.3OD): .delta. 8.16 (s, 1H), 7.47 (s,
1H), 3.99 (d, J=13.6 Hz, 1H), 3.95 (d, J=13.6 Hz, 1H), 3.68 (dd,
J=11.3, 4.8 Hz, 1H), 3.54 (dd, J=11.3, 6.5 Hz, 1H), 2.49 (m, 1H),
1.90 (m, 1H), 0.93 (d, J=6.9 Hz, 3H), 0.88 (d, J=6.9 Hz, 3H).
.sup.13C NMR (125.7 MHz, CD.sub.3OD, centre line .delta. 49.0):
.delta. 152.1 (C), 150.8 (CH), 146.7 (C), 129.0 (CH), 115.4 (C),
115.0 (C), 64.6 (CH.sub.2), 62.3 (CH), 41.9 (CH), 29.8 (CH.sub.2),
19.2 (CH.sub.3), 18.9 (CH.sub.3). ESI-HRMS calcd for
C.sub.12H.sub.20N.sub.5O.sup.+ (M+H).sup.+, 250.1663. found
250.1661.
Example H. Synthesis of
(2S)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-4-methylp-
entan-1-ol (H.1)
##STR00023##
[0058]
(2S)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-4-m-
ethylpentan-1-ol (H.1)
[0059] (2S)-2-Amino-4-methyl-pentan-1-ol (0.100 g, 0.85 mmol),
9-deazaadenine (0.114 g, 0.85 mmol), and aq. formaldehyde solution
(37%, 0.077 mL, 1.02 mmol) were stirred in tert-butanol (3 mL) at
70.degree. C. for 16 h. Silica gel was added to absorb all the
solvent then the solvent was evaporated and the residue purified by
chromatography on silica gel (CHCl.sub.3-7M NH.sub.3/MeOH, 93:7
then 85:15) to give H.1 as a colourless solid (0.102 g, 45%).
.sup.1H NMR (500 MHz, CD.sub.3OD): .delta. 8.16 (s, 1H), 7.47 (s,
1H), 3.97 (s, 2H), 3.65 (dd, J=11.3, 4.4 Hz, 1H), 3.47 (dd, J=11.3,
6.5 Hz, 1H), 2.75 (m, 1H), 1.62 (m, 1H), 1.35 (ddd, J=13.7, 7.4,
6.2 Hz, 1H), 1.28 (ddd, J=13.9, 7.2, 7.2 Hz, 1H), 0.88 (d, J=6.6
Hz, 3H), 0.80 (d, J=6.6 Hz, 3H). .sup.13C NMR (125.7 MHz,
CD.sub.3OD, centre line .delta. 49.0): .delta. 152.1 (C), 150.8
(CH), 146.6 (C), 129.0 (CH), 115.5 (C), 114.7 (C), 64.6 (CH.sub.2),
57.2 (CH), 41.6 (CH.sub.2), 41.0 (CH.sub.2), 26.0 (CH), 23.3
(CH.sub.3), 23.1 (CH.sub.3). ESI-HRMS calcd for
C.sub.13H.sub.22N.sub.5O.sup.+, (M+H).sup.+, 264.1819. found
264.1820.
Example I. Synthesis of
(2S,3S)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-meth-
ylpentan-1-ol (I.1)
##STR00024##
[0060]
(2S,3S)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]--
3-methylpentan-1-ol (I.1)
[0061] (2S,3S)-2-Amino-3-methylpentan-1-ol (0.120 g, 1.02 mmol),
9-deazaadenine (0.137 g, 1.02 mmol), and aq. formaldehyde solution
(37%, 0.092 mL, 1.22 mmol) were heated and stirred in tert-butanol
(3 mL) at 70.degree. C. for 16 h. Silica gel was added to absorb
all the solvent then the solvent was evaporated and the residue
purified by chromatography on silica gel (CHCl.sub.3-7M
NH.sub.3/MeOH, 92:8 then 89:11 then 85:15) to afford 1.1 as a
colourless waxy solid (0.120 g, 45%). .sup.1H NMR (500 MHz,
CD.sub.3OD): .delta. 8.16 (s, 1H), 7.47 (s, 1H), 3.98 (s, 2H), 3.69
(dd, J=11.3, 4.3 Hz, 1H), 3.51 (dd, J=11.3, 7.1 Hz, 1H), 2.62 (ddd,
J=7.0, 4.4, 4.4 Hz, 1H), 1.67 (m, 1H), 1.46 (m, 1H), 1.17 (m, 1H),
0.89-0.85 (m, 6H). .sup.13C NMR (125.7 MHz, CD.sub.3OD, centre line
.delta. 49.0): .delta. 152.1 (C), 150.8 (CH), 146.7 (C), 129.0
(CH), 115.4 (C), 114.9 (C), 63.1 (CH), 62.1 (CH.sub.2), 41.8
(CH.sub.2), 36.7 (CH), 27.2 (CH.sub.2), 15.0 (CH.sub.3), 12.3
(CH.sub.3). ESI-HRMS calcd for C.sub.13H.sub.22N.sub.5O.sup.+,
(M+H).sup.+, 264.1819. found 264.1820.
Example J. Synthesis of
(2S)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-2-phenyle-
than-1-ol (J.1)
##STR00025##
[0062]
(2S)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-2-p-
henylethan-1-ol (J1)
[0063] (2S)-2-Amino-2-phenyl-ethanol (0.150 g, 1.09 mmol)
9-deazaadenine (0.147 g, 1.10 mmol), and aq. formaldehyde solution
(37%, 0.098 mL, 1.31 mmol) were stirred and heated in tert-butanol
(3 mL) at 70.degree. C. for 16 h. Silica gel was added to absorb
all the solvent then the solvent was evaporated and the residue
purified by chromatography on silica gel (CHCl.sub.3-7M
NH.sub.3/MeOH, 92:8 then 89:11 then 85:15) to afford J.1 as a
colourless foam (0.117 g, 38%). .sup.1H NMR (500 MHz, CD.sub.3OD):
.delta. 8.14 (s, 1H), 7.38-7.31 (m, 5H), 7.28-7.24 (m, 1H),
3.88-3.82 (m, 2H), 3.75 (d, J=13.8 Hz, 1H), 3.65 (dd, J=11.0, 4.9
Hz, 1H), 3.60 (dd, J=11.0, 8.4 Hz, 1H). .sup.13C NMR (125.7 MHz,
CD.sub.3OD, centre line .delta. 49.0): .delta. 152.0 (C), 150.8
(CH), 146.6 (C), 141.5 (C), 129.6 (CH), 129.0 (CH), 128.8 (CH),
128.6 (CH), 115.5 (C), 114.7 (C), 67.7 (CH.sub.2), 65.4 (CH), 41.7
(CH.sub.2). ESI-HRMS calcd for C.sub.15H.sub.18N.sub.5O.sup.+,
(M+H).sup.+, 284.1506. found 284.1501.
Example K. Synthesis of
(2R)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-[(4-chl-
orophenyl)sulfanyl]propan-1-ol (K.3)
##STR00026##
[0064] tert-Butyl
(4R)-4-{[(4-chlorophenyl)sulfanyl]methyl}-2,2-dimethyl-1,3-oxazolidine-3--
carboxylate (K.1)
[0065] Step 1. Methanesulfonyl chloride (0.40 ml, 5.19 mmol) was
added dropwise to a stirred solution of tert-butyl
(4S)-4-(hydroxymethyl)-2,2-dimethyl-1,3-oxazolidine-3-carboxylate
[prepared as for its enantiomer, Dondoni, et al..sup.13] (1.00 g,
4.32 mmol) and triethylamine (1.22 ml, 8.65 mmol) in
CH.sub.2Cl.sub.2 (15 mL) at 0.degree. C. The mixture was warmed to
rt and stirred for 20 min then washed with sat NaHCO.sub.3
(3.times.5 mL), dried, and the solvent evaporated to give the crude
mesylate as an oil (1.22 g, 3.94 mmol).
Step 2
[0066] 4-chlorobenzene-1-thiol (0.351 g, 2.42 mmol) was added to a
solution of sodium hydride (60%, 0.089 g, 2.23 mmol) in DMF (3 mL)
at 0.degree. C. After 20 min a solution of the mesylate from step 1
above (0.30 g, 0.97 mmol) in DMF (0.75 ml) was added and the
mixture warmed to rt and stirred for 16 h. Water (2 mL) was added
and the mixture extracted with Et.sub.2O (60 mL). The extract was
washed with H.sub.2O (3.times.5 mL), brine (5 mL), dried and the
solvent evaporated to a colourless oil that was chromatographed on
silica gel (gradient of 0-6% EtOAc in hexanes) to give K.1 as a
colourless gum (0.234 g, 67%). [.alpha.].sub.D.sup.20 -17.9 (c
1.05, CHCl.sub.3). .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.
7.40-7.30 (m, 2H), 7.28-7.22 (m, 2H), 4.11-3.98 (m, 1.5H),
3.94-3.89 (m, 1.5H), 3.50 (d, J=13.7 Hz, 0.5H), 3.27 (d, J=13.4 Hz,
0.5H), 2.79 (dd, J=13.5, 10.7 Hz, 1H), 1.63-1.56 (m, 3H), 1.51-1.42
(m, 12H). .sup.13C NMR (125.7 MHz, CDCl.sub.3, centre line .delta.
77.0): .delta. 152.1, 151.4 (C), 134.2, 133.8 (C), 132.5, 131.6
(C), 130.9, 129.3 (CH), 129.1 (CH), 94.5, 93.9 (C), 80.5, 80.3 (C),
66.0 (CH.sub.2), 56.6, 56.3 (CH), 35.7, 33.8 (CH.sub.2), 28.5, 28.4
(CH.sub.3), 27.7, 27.0 (CH.sub.3), 24.3, 23.1 (CH.sub.3). ESI-HRMS
calcd for C.sub.17H.sub.2435ClNNaO.sub.3S.sup.+, (M+Na).sup.+,
380.1058. found 380.1056.
(2R)-2-Amino-3-[(4-chlorophenyl)sulfanyl]propan-1-ol hydrochloride
(K.2)
[0067] Compound K.1 (0.228 g, 0.64 mmol) was dissolved in MeOH (3
mL), cooled to 0.degree. C. and aq. hydrochloric acid (36%, 2 mL)
added. The mixture was stirred at rt for 16 h, then the solvent was
evaporated to give K.2 as a colourless solid (0.161 g, 99%).
[.alpha.].sub.D.sup.20 -27.7 (c 1.09, MeOH). .sup.1H NMR (500 MHz,
CD.sub.3OD): .delta. 7.47 (d, J=8.6 Hz, 2H), 7.36 (d, J=8.7 Hz,
2H), 3.82 (dd, J=11.7, 3.4 Hz, 1H), 3.74 (dd, J=11.7, 5.0 Hz, 1H),
3.30-3.25 (m, 2H), 3.19 (dd. J=16.3, 9.2 Hz, 1H). .sup.13C NMR
(125.7 MHz, CD.sub.3OD, centre line .delta. 49.0): .delta. 134.31
(C), 134.26 (C), 132.9 (CH), 130.5 (CH), 61.2 (CH.sub.2), 53.5
(CH), 34.1 (CH.sub.2). ESI-HRMS calcd for
C.sub.9H.sub.13.sup.35ClNOS.sup.+, (M-HCl+H).sup.+, 218.0401. found
218.0408.
(2R)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-[(4-chlo-
rophenyl)sulfanyl]propan-1-ol (K.3)
[0068] Compound K.2 (0.151 g, 0.59 mmol) was dissolved in MeOH (10
mL) and neutralized with Amberlyst A21 resin. The mixture was then
passed through a short column of the same resin and eluted with
MeOH to give the free amino form of K.2 as a yellow oil (129 mg).
This was dissolved in tert-butanol (3 mL) then aq. formaldehyde
solution (37%, 0.060 mL, 0.80 mmol) and 9-deazaadenine (0.080 g,
0.60 mmol) were added and the mixture stirred at 70.degree. C. for
16 h. Silica gel was added to absorb all the solvent then the
solvent was evaporated and the residue purified by chromatography
on silica gel (CHCl.sub.3-7M NH.sub.3/MeOH, 92:8 then 85:15) and
the fractions containing the product were evaporated. The residue
was further purified on silica gel (CHCl.sub.3-MeOH-28% aq.
NH.sub.4OH, 92:8:0.5) to give K.3 as a colourless foam (0.022 g,
10%). .sup.1H NMR (500 MHz, CD.sub.3OD): .delta. 8.16 (s, 1H), 7.35
(s, 1H), 7.13-7.08 (m, 4H), 4.02 (d, J=14.0 Hz, 1H), 3.92 (d,
J=14.0 Hz, 1H), 3.70 (dd, J=11.3, 5.2 Hz, 1H), 3.66 (dd, J=11.3,
5.2 Hz, 1H), 3.13 (dd, J=13.8, 6.2 Hz, 1H), 2.92 (dd, J=13.8, 6.9
Hz, 1H), 2.75 (m, 1H). .sup.13C NMR (125.7 MHz, CD.sub.3OD, centre
line .delta. 49.0): .delta. 152.0 (C), 150.8 (CH), 146.5 (C), 135.8
(C), 133.0 (C), 131.8 (CH), 129.8 (CH), 129.1 (CH), 115.5 (C),
114.4 (C), 63.5 (CH.sub.2), 57.0 (CH), 41.0 (CH.sub.2), 36.1
(CH.sub.2). ESI-HRMS calcd for
C.sub.16H.sub.18.sup.35ClN.sub.5NaOS.sup.+ (M+Na).sup.+, 386.0813.
found 386.0816.
Example L. Synthesis of
(2R)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-(benzyl-
sulfanyl)propan-1-ol (L.3)
##STR00027##
[0069] tert-Butyl
(4R)-4-[(benzylsulfanyl)methyl]-2,2-dimethyl-1,3-oxazolidine-3-carboxylat-
e (L.1)
[0070] Phenylmethanethiol (0.285 ml, 2.42 mmol) was added to a
stirred solution of sodium hydride (60%, 0.089 g, 2.23 mmol) in DMF
(3 mL) at 0.degree. C. After 20 min a solution of the crude
mesylate (0.3 g, 0.97 mmol, from step 1 of the preparation of K.1)
in DMF (0.75 ml) was added then the mixture was warmed to rt and
stirred for 16 h. Water (2 ml) was added and the mixture extracted
with Et.sub.2O (60 mL). The extract was washed with H.sub.2O
(3.times.5 mL), brine (5 mL), dried and the solvent evaporated to a
colourless oil that was chromatographed on silica gel (gradient of
0-6% EtOAc in hexanes) to give L.1 as a colourless gum (0.166 g,
51%). [.alpha.].sub.D.sup.20 +61.7 (c 1.12, CHCl.sub.3). .sup.1H
NMR (500 MHz, CDCl.sub.3): .delta. 7.40-7.28 (m, 4H), 7.26-7.20 (m,
1H), 4.09 (d, J=7.1 Hz, 0.5H), 3.99-3.85 (m, 2.5H). 3.76 (s, 2H),
2.86 (m, 1H), 2.51 (q, J=13.0 Hz, 1H), 1.58, 1.53, 1.49, 1.47,
1.46, 1.42 (6.times.s, 15H). .sup.13C NMR (125.7 MHz, CDCl.sub.3,
centre line .delta. 77.0): .delta. 152.0, 151.4 (C), 138.5, 138.1
(C), 129.0, 128.7 (CH), 128.6, 128.4 (CH), 127.2, 126.9 (CH), 94.2,
93.7 (C), 80.3, 79.9 (C), 66.5 (CH.sub.2), 57.2, 56.9 (CH), 36.9,
36.4 (CH.sub.2), 34.8, 33.6 (CH.sub.2), 28.4 (CH.sub.3), 27.6, 26.8
(CH.sub.3), 24.4, 23.2 (CH.sub.3). ESI-HRMS calcd for
C.sub.18H.sub.2735ClNNaO.sub.3S.sup.+ (M+Na).sup.+, 360.1604. found
360.1592.
(2R)-2-Amino-3-(benzylsulfanyl)propan-1-ol hydrochloride (L.2
[0071] Compound L.1 (0.166 g, 0.49 mmol) was dissolved in MeOH (3
mL), cooled to 0.degree. C. and aq. hydrochloric acid (36%, 2 mL)
added. After stirring at rt for 1.5 h, the solvent was evaporated
to a colourless gum that scratched down to a colourless solid
(0.115 g, 100%). [.alpha.].sub.D.sup.20 -52.6 (c 0.91, MeOH).
.sup.1H NMR (500 MHz, CD.sub.3OD): .delta. 7.38-7.35 (m, 2H),
7.34-7.30 (m, 2H), 7.25 (m, 1H), 3.81 (s, 2H), 3.77 (dd, J=11.7,
3.8 Hz, 1H), 3.65 (dd, J=11.7, 5.7, Hz, 1H), 3.27 (m, 1H), 2.74
(dd, J=14.2, 6.7 Hz, 1H), 2.67 (dd, J=14.2, 7.4 Hz, 1H). .sup.13C
NMR (125.7 MHz, CD.sub.3OD, centre line .delta. 49.0): .delta.
139.2 (C), 130.1 (CH), 129.7 (CH), 128.3 (CH), 61.5 (CH.sub.2),
53.7 (CH), 37.1 (CH.sub.2), 31.2 (CH.sub.2). ESI-HRMS calcd for
C.sub.10H.sub.1635ClNOS.sup.+, (M-HCl+H).sup.+, 198.0948. found
198.0948.
(2R)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-(benzyls-
ulfanyl)propan-1-ol (L.3)
[0072] Compound L.2 (0.170 g, 0.73 mmol) was dissolved in MeOH (10
mL) and neutralized with Amberlyst A21 resin. The mixture was then
passed through a small column of the same resin and eluted with
MeOH to give the free amino form of L.2 as a yellow oil. This was
dissolved in tert-butanol (3 mL) then aq. formaldehyde solution
(37%, 0.071 mL, 0.95 mmol) and 9-deazaadenine (0.098 g, 0.73 mmol)
added and the mixture was stirred at 70.degree. C. for 16 h. Silica
gel was added to absorb all the solvent then the solvent was
evaporated and the residue purified by chromatography on silica gel
(CHCl.sub.3-MeOH-28% aq. NH4OH, 92:8:0.5). The fractions containing
the product were evaporated and the residue was further purified on
silica gel (gradient of 2-10% 7M NH.sub.3/MeOH--CHCl.sub.3) to give
L.3 as a colourless solid (0.021 g, 8%). .sup.1H NMR (500 MHz,
CD.sub.3OD): .delta. 8.17 (s, 1H), 7.43 (s, 1H), 7.25-7.21 (m, 2H),
7.19-7.15 (m, 3H), 3.99 (d, J=13.8 Hz, 1H), 3.91 (d, J=13.8 Hz,
1H), 3.66 (dd, J=11.2, 4.9 Hz, 1H), 3.57 (dd, J=11.2, 5.5 Hz, 1H),
3.53 (d, J=1.6 Hz, 2H), 2.77 (m, 1H), 2.60 (dd, J=13.6, 6.4 Hz,
1H), 2.49 (dd, J=13.6, 6.9 Hz, 1H). .sup.13C NMR (125.7 MHz,
CD.sub.3OD, centre line .delta. 49.0): .delta. 152.1 (C), 150.9
(CH), 146.6 (C), 139.8 (C), 129.9 (CH), 129.4 (CH), 129.1 (CH),
127.9 (CH), 115.5 (C), 114.7 (C), 63.8 (CH.sub.2), 57.8 (CH), 41.2
(CH.sub.2), 36.9 (CH.sub.2), 33.9 (CH.sub.2). ESI-HRMS calcd for
C.sub.17H.sub.21N.sub.5NaOS.sup.+ (M+Na).sup.+, 366.1360. found
366.1362.
Example P. Synthesis of
(2S)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-(1H-imi-
dazol-4-yl)propan-1-ol (P.4)
##STR00028##
[0073]
(4S)-4-{[1-(Triphenylmethyl)-1H-imidazol-4-yl]methyl}-1,3-oxazolidi-
n-2-one or
(4S)-4-{[1-(triphenylmethyl)-1H-imidazol-5-yl]methyl}-1,3-oxazo-
lidin-2-one (P.1)
[0074] A modified literature procedure [Madrigal, et al..sup.14]
was followed. (S)-Histidinol dihydrochloride (0.500 g, 2.34 mmol)
and diethyl carbonate (2.86 mL, 23.61 mmol) were stirred together
in ethanol (24 mL), then sodium methoxide in methanol solution
(25%, 1.6 mL, 7.0 mmol) added. The mixture was heated under reflux
for 72 h then the solvent was evaporated and the residue
chromatographed on silica gel (CHCl.sub.3-MeOH-28% aq. NH.sub.4OH,
9:1:0.1) to give
(4S)-4-(1H-imidazol-4-ylmethyl)-1,3-oxazolidin-2-one as a
colourless solid (0.26 g, 1.56 mmol, 90-95% pure). .sup.1H NMR (500
MHz, CD.sub.3OD): .delta. 7.61 (d, J=1.0 Hz, 1H), 6.93 (s, 1H),
4.45-4.40 (m, 1H), 4.18-4.12 (m, 2H), 2.86 (dd, J=14.7, 4.8 Hz,
1H), 2.80 (dd, J=14.8, 6.1 Hz, 1H). It was dissolved in DMF (4 mL)
then triethylamine (0.42 mL, 3.00 mmol) and trityl chloride (0.489
g, 1.70 mmol) were added. The mixture was stirred for 60 h at rt
then diluted with Et.sub.2O (60 mL) and the mixture washed with
H.sub.2O (4.times.5 mL), brine (5 mL), dried and the solvent
evaporated. The residue was chromatographed on silica gel (gradient
of 0-5% MeOH in EtOAc) to give P.1 as a colourless foam (0.520 g,
54%). .sup.1H NMR (500 MHz, CD.sub.3OD): .delta. 7.41 (d, J=1.4 Hz,
1H), 7.39-7.34 (m, 9H), 7.16-7.11 (m, 6H), 6.82 (m, 1H), 4.39 (t,
J=8.4 Hz, 1H), 4.19-4.11 (m, 2H), 2.78 (dd, J=14.6, 4.9 Hz, 1H),
2.73 (dd, J=14.6, 6.1 Hz, 1H). .sup.13C NMR (125.7 MHz, CD.sub.3OD,
centre line .delta. 49.0): .delta. 162.1 (C), 143.6 (C.times.3),
139.8 (CH), 136.6 (C), 130.9 (CH), 129.32 (CH), 129.27 (CH), 121.6
(CH), 76.9 (C), 70.4 (CH.sub.2), 53.4 (CH), 33.9 (CH.sub.2).
ESI-HRMS calcd for C.sub.26H.sub.23N.sub.3NaO.sub.2.sup.+,
(M+Na).sup.+, 432.1683. found 432.1677.
(2S)-2-Amino-3-[1-(triphenylmethyl)-1H-imidazol-4-yl]propan-1-ol or
(2S)-2-amino-3-[1-(triphenylmethyl)-1H-imidazol-5-yl]propan-1-ol
(P.2)
[0075] Compound P.1 (0.510 g, 1.25 mmol) was dissolved in
2-propanol (7 mL) and potassium hydroxide (2 M, 3 mL, 6 mmol)
added. The mixture was heated at 80.degree. C. for 6 h then silica
gel was added to absorb all the solvent then the solvent was
evaporated and the residue chromatographed on silica gel
(CHCl.sub.3-MeOH-28% aq. NH.sub.4OH, 9:1:0.1) to give P.2 as a
colourless gum (0.478 g, 100%). .sup.1H NMR (500 MHz, CD.sub.3OD):
.delta. 7.40 (d, J=1.4 Hz, 1H), 7.38-7.34 (m, 9H), 7.18-7.13 (m,
6H), 6.75 (m, 1H), 3.52 (dd, J=10.9, 4.5 Hz, 1H), 3.35 (dd, J=10.9,
6.8 Hz, 1H), 3.09-3.03 (m, 1H), 2.65 (dd, J=14.4, 6.0 Hz, 1H), 2.50
(dd, J=14.4, 7.4 Hz, 1H). .sup.13C NMR (125.7 MHz, CD.sub.3OD,
centre line .delta. 49.0): .delta. 143.7 (C), 139.7 (CH), 139.2
(C), 130.8 (CH), 129.3 (CH), 129.2 (CH), 121.0 (CH), 76.8 (C), 66.7
(CH.sub.2), 53.8 (CH), 33.0 (CH.sub.2). ESI-HRMS calcd for
C.sub.25H.sub.26N.sub.3O.sup.+, (M+H).sup.+, 384.2071. found
384.2068.
[0076]
(2S)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-[-
1-(triphenylmethyl)-1H-imidazol-4-yl]propan-1-ol or
(2S)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-[1-(tri-
phenylmethyl)-1H-imidazol-5-yl]propan-1-ol (P.3). Compound P.2
(0.200 g, 0.52 mmol), 9-deazaadenine (0.070 g, 0.52 mmol) and aq.
formaldehyde solution (37%, 0.051 mL, 0.68 mmol) were heated at
70.degree. C. in tert-butanol (3 mL) for 16 h. Silica gel was added
to absorb all the solvent then the solvent was evaporated and the
residue chromatographed on silica gel (10% 7M NH3/MeOH--CHCl.sub.3)
to give P.3 as a colourless foam (0.101 g, 37%). .sup.1H NMR (500
MHz, CD.sub.3OD): .delta. 8.03 (s, 1H), 7.39 (s, 1H), 7.35 (d,
J=1.3 Hz, 1H), 7.33-7.29 (m, 9H), 7.12-7.08 (m, 6H), 6.75 (d, J=1.2
Hz, 1H), 3.96 (d, J=13.7 Hz, 1H), 3.93 (d, J=13.6 Hz, 1H), 3.61
(dd, J=11.3, 4.8 Hz, 1H), 3.49 (dd, J=11.3, 6.1 Hz, 1H), 3.02-2.97
(m, 1H), 2.72 (dd, J=14.5, 6.5 Hz, 1H), 2.69 (dd, J=14.5, 6.7 Hz,
1H). .sup.13C NMR (125.7 MHz, CD.sub.3OD, centre line .delta.
49.0): .delta. 152.0 (C), 150.8 (CH), 146.6 (C), 143.7 (C.times.3),
139.5 (CH), 139.2 (C), 130.8 (CH), 129.24 (CH), 129.20 (CH), 128.9
(CH), 121.2 (CH), 115.4 (C), 114.8 (C), 76.8 (C), 64.3 (CH.sub.2),
59.4 (CH), 41.3 (CH.sub.2), 30.7 (CH.sub.2). ESI-HRMS calcd for
C.sub.32H.sub.32N.sub.7O.sup.+, (M+H).sup.+, 530.2663. found
530.2666.
(2S)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-(1H-imid-
azol-4-yl)propan-1-ol (P.4)
[0077] Trifluoroacetic acid (0.6 mL, 8 mmol) was added to a stirred
solution of P.3 (0.100 g, 0.19 mmol) and triethylsilane (0.090 mL,
0.57 mmol) in CH.sub.2Cl.sub.2 (3 mL). After 2 h, the solvent was
evaporated and the residue dissolved in MeOH and the solvent
evaporated (3.times.). The residue was again dissolved in MeOH,
silica gel added and the solvent evaporated. Flash chromatography
on silica gel (CHCl.sub.3-MeOH-28% aq. NH.sub.4OH, 7:2.5:0.5) gave
P.4 as a colourless gum which crystallized on standing (0.050 g,
92%). .sup.1H NMR (500 MHz, CD.sub.3OD): .delta. 8.14 (s, 1H), 7.52
(d, J=0.9 Hz, 1H), 7.42 (s, 1H), 6.80 (s, 1H), 4.01 (d, J=13.6 Hz,
1H), 3.98 (d, J=13.6 Hz, 1H), 3.63 (dd, J=11.3, 4.7 Hz, 1H), 3.50
(dd, J=11.3, 5.9 Hz, 1H), 3.01-2.96 (m, 1H), 2.76 (d, J=6.7 Hz,
2H). .sup.13C NMR (125.7 MHz, CD.sub.3OD, centre line .delta.
49.0): .delta. 152.0 (C), 150.8 (CH), 146.6 (C), 136.1 (CH), 134.8
(b, C), 129.0 (CH), 119.4 (b, CH), 115.4 (C), 114.3 (C), 64.0
(CH.sub.2), 59.3 (CH), 41.3 (CH.sub.2), 29.2 (CH.sub.2). ESI-HRMS
calcd for C.sub.13H.sub.18N.sub.7O.sup.+, (M+H).sup.+, 288.1568.
found 288.1567.
Example Q. Synthesis of
(2R)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-(butyls-
ulfanyl)propan-1-ol (Q.2)
##STR00029##
[0078] tert-Butyl
(4R)-4-[(butylsulfanyl)methyl]-2,2-dimethyl-1,3-oxazolidine-3-carboxylate
(Q.1
[0079] Sodium hydride (60%, 0.097 g, 2.4 mmol) was added in
portions to a solution of butane-1-thiol (0.53 mL, 4.85 mmol) in
DMF (5 mL). After 15 min a solution of the crude mesylate (0.500 g,
1.62 mmol, from step 1 of the preparation of K.1) in DMF (1 mL) was
added. The mixture was stirred for 16 h then H.sub.2O (6 mL) added.
After extraction with Et.sub.2O (100 mL), the extract was washed
with H.sub.2O (4.times.5 mL), brine (5 mL), dried and the solvent
evaporated. The residue was chromatographed on silica gel (gradient
of 0-10% EtOAc in hexanes) to give Q.1 as a colourless oil (0.429
g, 88%). .sup.1H NMR (500 MHz, CDCl.sub.3): .delta. 4.06-3.92 (m,
2.5H), 3.91-3.85 (m, 0.5H), 2.94 (d, J=13.0 Hz, 0.5H), 2.80 (d,
J=13.0 Hz, 0.5H), 2.62-2.47 (m, 3H), 1.65-1.53 (m, 5H), 1.50-1.35
(m, 14H), 0.91 (t, J=7.1 Hz, 3H). .sup.13C NMR (125.7 MHz,
CDCl.sub.3, centre line .delta. 77.0): .delta. 152.1, 151.4 (C),
94.3, 93.7 (C), 80.2, 79.9 (C), 66.5, 66.3 (CH.sub.2), 57.4, 57.2
(CH), 34.6, 33.9 (CH.sub.2), 32.0, 31.9, (2.times.CH.sub.2), 28.5,
28.4 (CH.sub.3), 27.7, 26.9 (CH.sub.3), 24.5, 23.2 (CH.sub.3), 21.9
(CH.sub.2), 13.6 (CH.sub.3). ESI-HRMS calcd for
C.sub.15H.sub.29NNaO.sub.3S.sup.+, (M+Na).sup.+, 326.1761. found
326.1760.
(2R)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-(butylsu-
lfanyl)propan-1-ol (Q.2)
[0080] Compound Q.1 (0.400 g, 1.32 mmol) was dissolved in MeOH (4
mL) and aq. hydrochloric acid (36%, 1 mL) was added. After 15 min
the solvent was evaporated and the resulting gum dissolved in MeOH
(10 mL) and neutralized with Amberlyst A21 resin then passed
through a short column of the same resin and eluted with MeOH. The
fractions containing product were evaporated to an oily residue
that was dissolved in tert butanol (4 mL) then 9-deazaadenine
(0.177 g, 1.32 mmol) and aq. formaldehyde solution (37%, 0.12 mL,
1.60 mmol) added and the mixture stirred at 70.degree. C. for 16 h.
Silica gel was added to absorb all the solvent then the solvent was
evaporated and the residue chromatographed on silica gel (gradient
of 5-15% 7M NH.sub.3/MeOH in CHCl.sub.3) to give Q.2 as a
colourless solid (0.131 g, 32%). .sup.1H NMR (500 MHz, CD.sub.3OD):
.delta. 8.16 (s, 1H), 7.49 (s, 1H), 4.06 (d, J=13.8 Hz, 1H), 3.97
(d, J=13.9 Hz, 1H), 3.69 (dd, J=11.2, 5.1 Hz, 1H), 3.63 (dd,
J=11.2, 5.4 Hz, 1H), 2.81-2.76 (m, 1H), 2.69 (dd, J=13.5, 6.3 Hz,
1H), 2.53 (dd, J=13.5, 6.9 Hz, 1H), 2.31 (ddd, J=12.5, 8.0, 6.5 Hz,
1H), 2.25 (ddd, J=12.5, 8.1, 6.7 Hz, 1H), 1.45-1.35 (m, 2H),
1.33-1.25 (m, 2H), 0.85 (t, J=7.3 Hz, 3H). .sup.13C NMR (125.7 MHz,
CD.sub.3OD, centre line .delta. 49.0): .delta. 152.1 (C), 150.9
(CH), 146.6 (C), 129.1 (CH), 115.5 (C), 114.6 (C), 63.9 (CH.sub.2),
57.8 (CH), 41.2 (CH.sub.2), 34.5 (CH.sub.2), 32.7 (CH.sub.2), 32.6
(CH.sub.2), 22.9 (CH.sub.2), 13.9 (CH.sub.3). ESI-HRMS calcd for
C.sub.14H.sub.24N.sub.5OS.sup.+, (M+H).sup.+, 310.1697. found
310.1702.
Example R. Synthesis of
(2R)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-(heptyl-
sulfanyl)propan-1-ol (R.2)
##STR00030##
[0081] tert-Butyl
(4R)-4-[(heptylsulfanyl)methyl]-2,2-dimethyl-1,3-oxazolidine-3-carboxylat-
e (R.1)
[0082] Sodium hydride (60%, 0.162 g, 4.05 mmol) was added in
portions to a solution of heptane-1-thiol (0.641 g, 4.85 mmol) in
DMF (5 mL) at rt. After 15 min, a solution of the crude mesylate
(0.500 g, 1.62 mmol, from step 1 of the preparation of K.1) in DMF
(1 mL) was added. The mixture was stirred for 16 h then H.sub.2O (6
mL) added. After extraction with Et.sub.2O (100 mL), the extract
was washed with H.sub.2O (4.times.5 mL), brine (5 mL) dried and
evaporated to an oily residue that was chromatographed on silica
gel (gradient of 0-12% EtOAc in hexanes) to give R.1 as a
colourless oil (0.443 g, 79%). .sup.1H NMR (500 MHz, CDCl.sub.3):
.delta. 4.05-3.98 (m, 1.5H), 3.97-3.93 (m, 1H), 3.91-3.85 (m,
0.5H), 2.93 (d, J=13.5 Hz, 0.5H), 2.80 (d, J=12.9 Hz, 0.5H),
2.62-2.45 (m, 3H), 1.63-1.53 (m, 5H), 1.51-1.43 (m, 12H), 1.41-1.22
(m, 8H), 0.88 (t, J=6.8 Hz, 3H). .sup.13C NMR (125.7 MHz,
CDCl.sub.3, centre line .delta. 77.0): .delta. 152.1, 151.5 (C),
94.3, 93.7 (C), 80.2, 79.9 (C), 66.5, 66.3 (CH.sub.2), 57.4, 57.2
(CH), 34.7, 33.8 (CH.sub.2), 32.4, 31.7 (2.times.CH.sub.2), 30.0,
29.8 (CH.sub.2), 28.9, 28.8, (2.times.CH.sub.2), 28.5, 28.4
(CH.sub.3), 27.7, 26.9 (CH.sub.3), 24.5, 23.2 (CH.sub.3), 22.6
(CH.sub.2), 14.0 (CH.sub.3). ESI-HRMS calcd for
C.sub.18H.sub.35NNaO.sub.3S.sup.+, (M+Na).sup.+, 368.2230. found
368.2227.
[0083]
(2R)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-(-
heptylsulfanyl)propan-1-ol (R.2). Compound R.1 (0.420 g, 1.22 mmol)
was dissolved in MeOH (4 mL) and aq. hydrochloric acid (36%, 1 mL)
added. After 15 min the solvent was evaporated to a gum that was
dissolved in a 7:3 mixture of MeOH--CHCl.sub.3 (10 mL) and
neutralized with Amberlyst A21 resin then passed through a short
column of the same resin and eluted with a 7:3 mixture of
MeOH--CHCl.sub.3. The fractions containing product were evaporated
to an oily residue that was dissolved in tert-butanol (4 mL) then
9-deazaadenine (0.130 g, 0.97 mmol) and aq. formaldehyde solution
(37%, 0.087 mL, 1.16 mmol) were added and the mixture stirred at
70.degree. C. for 16 h. Silica gel was added to absorb all the
solvent then the solvent was evaporated and the residue
chromatographed on silica gel (gradient of 5-15% 7M NH.sub.3/MeOH
in CHCl.sub.3) to give crude R.2 (210 mg). Further chromatography
on silica gel (gradient of 0-5% aq. NH.sub.4OH (28%) in 2-PrOH)
gave R.2 as a colourless solid (0.134 g, 34%). .sup.1H NMR (500
MHz, CD.sub.3OD): .delta. 8.16 (s, 1H), 7.49 (s, 1H), 4.06 (d,
J=13.8 Hz, 1H), 3.97 (d, J=13.8 Hz, 1H), 3.69 (dd, J=11.2, 5.1 Hz,
1H), 3.63 (dd, J=11.2, 5.4 Hz, 1H), 2.81-2.76 (m, 1H), 2.69 (dd,
J=13.5, 6.3 Hz, 1H), 2.54 (dd, J=13.5, 6.9 Hz, 1H), 2.32 (ddd,
J=12.5, 7.9, 6.8 Hz, 1H), 2.26 (ddd, J=12.6, 7.9, 6.9 Hz, 1H),
1.47-1.37 (m, 2H), 1.33-1.20 (m, 8H), 0.88 (t, J=7.1 Hz, 3H).
.sup.13C NMR (125.7 MHz, CD.sub.3OD, centre line .delta. 49.0):
.delta. 152.1 (C), 150.9 (CH), 146.6 (C), 129.1 (CH), 115.4 (C),
114.6 (C), 63.8 (CH.sub.2), 57.9 (CH), 41.2 (CH.sub.2), 34.5
(CH.sub.2), 32.9 (CH.sub.2.times.2), 30.6 (CH.sub.2), 30.0
(CH.sub.2), 29.8 (CH.sub.2), 26.6 (CH.sub.2), 14.4 (CH.sub.3).
ESI-HRMS calcd for C.sub.17H.sub.30N.sub.5OS.sup.+, (M+H).sup.+,
352.2166. found 352.2157.
Example S. Synthesis of
(2S)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]heptan-1-o-
l (S.2)
##STR00031##
[0084] tert-Butyl
(4S)-2,2-dimethyl-4-pentyl-1,3-oxazolidine-3-carboxylate (S.1)
[0085] tert-Butyl
(4S)-2,2-dimethyl-4-(3-oxopropyl)-1,3-oxazolidine-3-carboxylate
[Goswami, et al..sup.15] (0.400 g, 1.55 mmol, purified on silica
gel with a gradient of 0-50% EtOAc in hexanes) and
ethyltriphenylphosphonium bromide [Pale{hacek over (c)}ek, J. et
al..sup.16] (0.866 g, 2.33 mmol, dried over P.sub.2O.sub.5 then
evaporated 2.times. from dry toluene) were dissolved in anhydrous
CH.sub.2Cl.sub.2 (7 mL) and cooled at 0.degree. C. A solution of
potassium tert-butoxide in THF (1.6 M, 1.5 mL, 2.4 mmol) was added
dropwise and the mixture stirred for 20 min. [method similar to
that described in Ksander, et al..sup.17] Silica gel was added to
absorb all the solvent then the solvent was evaporated and the
residue chromatographed on silica gel (gradient of 0-5% EtOAc in
hexanes to give the Wittig product as a colourless oil (0.304 g,
73%). The latter product (0.430 g, 1.60 mmol) and 10% Pd on carbon
(60 mg) were stirred together in EtOAc (15 mL) under a hydrogen
atmosphere for 16 h. The mixture was filtered through Celite then
the filtrate evaporated and the residue chromatographed on silica
gel (gradient of 0-5% EtOAc in hexanes) to give S.1 as a colourless
oil (0.359 g, 61%). .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.
3.93-3.71 (m, 3H), 1.83-1.42 (m, 17H), 1.36-1.20 (m, 6H), 0.93-0.80
(m, 3H). .sup.13C NMR (125.7 MHz, CDCl.sub.3, centre line .delta.
77.0): .delta. 152.1, 151.9 (C), 93.6, 93.0 (C), 79.8, 79.3 (C),
67.1, 66.8 (CH.sub.2), 57.8, 57.4 (CH), 33.6, 32.8 (CH.sub.2), 31.7
(CH.sub.2), 28.5 (CH.sub.3), 27.5, 26.7 (CH.sub.3), 25.9
(CH.sub.2), 24.6, 23.3 (CH.sub.3), 22.6 (CH.sub.2), 13.9
(CH.sub.3). ESI-HRMS calcd for C.sub.15H.sub.29NNaO.sub.3.sup.+,
(M+Na).sup.+, 294.2040. found 294.2038.
(2S)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]heptan-1-ol
(S.2)
[0086] Compound S.1 (0.358 g, 1.32 mmol) was dissolved in MeOH (4
mL) and aq. hydrochloric acid (36%, 1 mL) added. After 15 min the
solvent was evaporated to a colourless solid that was dissolved in
MeOH (10 mL), neutralized with Amberlyst A21 resin then passed
through a short column of the same resin and eluted with MeOH. The
fractions containing product were evaporated to an oily residue
that was dissolved in tert-butanol (4 mL) then 9-deazaadenine
(0.177 g, 1.32 mmol) and aq. formaldehyde solution (0.12 mL, 1.59
mmol) were added and the mixture stirred at 70.degree. C. for 16 h.
Silica gel was added to absorb all the solvent then the solvent was
evaporated and the residue chromatographed on silica gel (gradient
of 5-15% 7M NH.sub.3/MeOH in CHCl.sub.3) to give S.2 as a
colourless solid (0.122 g, 33%). .sup.1H NMR (500 MHz, CD.sub.3OD):
.delta. 8.16 (s, 1H), 7.47 (s, 1H), 3.64 (dd, J=11.2, 4.5 Hz, 1H),
3.48 (dd, J=11.2, 6.5 Hz, 1H), 2.68-2.64 (m, 1H), 1.52-1.38 (m,
2H), 1.32-1.17 (m, 6H), 0.86 (t, J=7.1 Hz, 3H). .sup.13C NMR (125.7
MHz, CD.sub.3OD, centre line .delta. 49.0): .delta. 152.1 (C),
150.8 (CH), 146.6 (C), 129.0 (CH), 115.4 (C), 114.8 (C), 64.4
(CH.sub.2), 59.2 (CH), 41.2 (CH.sub.2), 33.1 (CH.sub.2), 32.0
(CH.sub.2), 26.8 (CH.sub.2), 23.6 (CH.sub.2), 14.4 (CH.sub.3).
ESI-HRMS calcd for C.sub.14H.sub.24N.sub.5O.sup.+, (M+H).sup.+,
278.1976. found 278.1974.
Example T. Synthesis of
(2S)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]octan-1-ol
(T.2)
##STR00032##
[0087] tert-Butyl
(4S)-4-hexyl-2,2-dimethyl-1,3-oxazolidine-3-carboxylate (T.1)
[0088] tert-Butyl
(4S)-2,2-dimethyl-4-(3-oxopropyl)-1,3-oxazolidine-3-carboxylate
[Goswami, et al..sup.15] (0.500 g, 1.94 mmol, purified on silica
gel with a gradient of 0-50% EtOAc in hexanes) and
n-propyltriphenylphosphonium bromide (1.12 g, 2.91 mmol, dried over
P.sub.2O.sub.5 then evaporated 2.times. from dry toluene) were
dissolved in anhydrous CH.sub.2Cl.sub.2 (7 mL) and cooled to
0.degree. C. A solution of potassium tert-butoxide in THF (1.6 M,
1.8 mL, 2.90 mmol) was added dropwise and the mixture stirred for
20 min. [method similar to that described in Ksander, et
al..sup.17] Silica gel was added to absorb all the solvent then the
solvent was evaporated and the residue chromatographed on silica
gel (gradient of 0-4% EtOAc in hexanes to give the Wittig product
as a colourless oil (0.361 g, 66%). The latter product (0.360 g,
1.27 mmol) and 10% Pd on carbon (60 mg) were stirred together in
EtOAc (15 mL) under a hydrogen atmosphere for 16 h. The mixture was
filtered through Celite then the filtrate evaporated and the
residue chromatographed on silica gel (gradient of 0-5% EtOAc in
hexanes) to give T.1 as a colourless oil (0.342 g, 94%). .sup.1H
NMR (500 MHz, CDCl.sub.3): .delta. 3.93-3.71 (m, 3H), 1.82-1.42 (m,
17H), 1.36-1.19 (m, 8H), 0.92-0.85 (m, 3H). .sup.13C NMR (125.7
MHz, CDCl.sub.3, centre line .delta. 77.0): .delta. 152.1, 151.9
(C), 93.5, 93.0 (C), 79.8, 79.3 (C), 67.1, 66.7 (CH.sub.2), 57.8,
57.4 (CH), 33.6, 32.9 (CH.sub.2), 31.8 (CH.sub.2), 29.1 (CH.sub.2),
28.5 (CH.sub.3), 27.5, 26.7 (CH.sub.3), 26.2 (CH.sub.2), 24.6, 23.3
(CH.sub.3), 22.5 (CH.sub.2), 14.0 (CH.sub.3). ESI-HRMS calcd for
C.sub.16H.sub.32NO.sub.3+, (M+H).sup.+, 286.2377. found
286.2375.
(2S)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]octan-1-ol
(T.2)
[0089] Compound T.1 (0.320 g, 1.12 mmol) was dissolved in MeOH (4
mL) and aq. hydrochloric acid (36%, 1 mL) added. After 15 min the
solvent was evaporated to a colourless solid that was dissolved in
a 4:1 mixture MeOH--CHCl.sub.3 (10 mL), neutralized with Amberlyst
A21 resin then passed through a short column of the same resin and
eluted with 4:1 MeOH--CHCl.sub.3. The fractions containing product
were evaporated to a yellow solid that was dissolved in
tert-butanol (4 mL) then 9-deazaadenine (0.150 g, 1.12 mmol) and
aq. formaldehyde solution (37%, 0.101 mL, 1.35 mmol) were added and
the mixture was stirred at 70.degree. C. for 16 h. Silica gel was
added to absorb all the solvent then the solvent was evaporated and
the residue chromatographed on silica gel (gradient of 5-15% 7M
NH.sub.3/MeOH in CHCl.sub.3) to give T.2 as a colourless solid
(0.148 g, 45%). .sup.1H NMR (500 MHz, 1:1 CD.sub.3OD-CDCl.sub.3):
.delta. 8.20 (s, 1H), 7.40 (s, 1H), 3.98 (d, J=13.7 Hz, 1H), 3.95
(d, J=13.7 Hz, 1H), 3.73 (dd, J=11.4, 4.0 Hz, 1H), 3.50 (dd,
J=11.4, 6.6 Hz, 1H), 2.74-2.69 (m, 1H), 1.54-1.39 (m, 2H),
1.34-1.22 (m, 8H), 0.88 (t, J=6.9 Hz, 3H). .sup.13C NMR (125.7 MHz,
1:1 CD.sub.3OD-CDCl.sub.3, centre lines .delta. 49.0 and .delta.
78.3): .delta. 151.2 (C), 150.2 (CH), 146.0 (C), 128.1 (CH), 115.0
(C), 114.2 (C), 63.6 (CH.sub.2), 59.0 (CH), 40.7 (CH.sub.2), 32.4
(CH.sub.2), 31.6 (CH.sub.2), 30.0 (CH.sub.2), 26.7 (CH.sub.2), 23.1
(CH.sub.2), 14.3 (CH.sub.3). ESI-HRMS calcd for
C.sub.15H.sub.25N.sub.5NaO.sup.+, (M+Na).sup.+, 314.1952. found
314.1953.
Example U. Synthesis of
(2S)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]undecan-1--
ol (U.2)
##STR00033##
[0090] tert-Butyl (4S)-2, 2-dimethyl-4-nonyl-1,
3-oxazolidine-3-carboxylate (U.1). Step 1
[0091] Triphenylphosphine (3.70 g, 14.11 mmol) and 1-bromohexane
(2.97 mL, 21.16 mmol) were stirred and heated under reflux in
toluene (10 mL) for 16 h. The resulting n-hexyltriphenylphosphonium
bromide (3.30 g, 55%) was filtered off and dried over
P.sub.2O.sub.5 then evaporated 2.times. from dry toluene.
Step 2
[0092] The phosphonium salt from step 1 (1.30 g, 3.04 mmol) and
tert-butyl
(4S)-2,2-dimethyl-4-(3-oxopropyl)-1,3-oxazolidine-3-carboxylate
[Goswami, et al..sup.15] (0.500 g, 1.94 mmol, purified on silica
gel with a gradient of 0-50% EtOAc in hexanes) were dissolved in
CH.sub.2Cl.sub.2 (7 mL) and cooled at 0.degree. C. A solution of
potassium tert-butoxide in THF (1.6 M, 1.9 mL, 3.00 mmol) was added
dropwise and the mixture stirred for 20 min. [method similar to
that described in Ksander, et al..sup.17]. Silica gel was added to
absorb all the solvent then the solvent was evaporated and the
residue chromatographed on silica gel (gradient of 0-5% EtOAc in
hexanes to give the Wittig product as a colourless oil (0.370 g,
59%). The latter product (0.340 g, 1.04 mmol) and 10% Pd on carbon
(60 mg) were stirred together in EtOAc (15 mL) under a hydrogen
atmosphere for 16 h. The mixture was filtered through Celite and
the filtrate evaporated and the residue chromatographed on silica
gel (gradient of 0-5% EtOAc in hexanes) to give U.1 as a colourless
oil (0.340 g, 99%). .sup.1H NMR (500 MHz, CDCl.sub.3): .delta.
3.93-3.70 (m, 3H), 1.82-1.42 (m, 17H), 1.35-1.82 (m, 14H), 0.88 (t,
J=6.9 Hz, 3H). .sup.13C NMR (125.7 MHz, CDCl.sub.3, centre line
.delta. 77.0): .delta. 152.2, 151.9 (C), 93.6, 93.0 (C), 79.8, 79.3
(C), 67.1, 66.8 (CH.sub.2), 57.8, 57.5 (CH), 33.6, 32.9 (CH.sub.2),
31.9 (CH.sub.2), 29.6, 29.5, 29.3 (4.times.CH.sub.2), 28.5
(CH.sub.3), 27.5, 26.8 (CH.sub.3), 26.3 (CH.sub.2), 24.6, 23.3
(CH.sub.3), 22.6 (CH.sub.2), 14.1 (CH.sub.3). ESI-HRMS calcd for
C.sub.19H.sub.37NNaO.sub.3.sup.+, (M+Na).sup.+, 350.2666. found
350.2663.
(2S)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]undecan-1-o-
l (U.2)
[0093] Compound U.1 (0.310 g, 0.947 mmol) was dissolved in MeOH (4
mL) and aq. hydrochloric acid (36%, 1 mL) added. After 15 min the
solvent was evaporated to a colourless solid that was dissolved in
a 4:1 mixture MeOH--CHCl.sub.3, neutralized with Amberlyst A21
resin then passed through a short column of the same resin and
eluted with 4:1 MeOH--CHCl.sub.3. The fractions containing product
were evaporated to a yellow oil that was dissolved in tert-butanol
(4 mL) then 9-deazaadenine (0.127 g, 0.95 mmol) and aq.
formaldehyde solution (37%, 0.085 mL, 1.10 mmol) were added and the
mixture was stirred at 70.degree. C. for 16 h. Silica gel was added
to absorb all the solvent then the solvent was evaporated and the
residue chromatographed on silica gel (gradient of 5-15% 7M
NH.sub.3/MeOH in CHCl.sub.3) to give U.2 as a colourless solid
(0.135 g, 43%). .sup.1H NMR (500 MHz, 1:1 CD.sub.3OD-CDCl.sub.3):
.delta. 8.20 (s, 1H), 7.40 (s, 1H), 3.96 (s, 1H), 3.73 (dd, J=11.4,
3.9 Hz, 1H), 3.50 (dd, J=11.4, 6.6 Hz, 1H), 2.74-2.69 (m, 1H),
1.55-1.39 (m, 2H), 1.35-1.21 (m, 14H), 0.89 (t, J=7.0 Hz, 3H).
.sup.13C NMR (125.7 MHz, 1:1 CD.sub.3OD-CDCl.sub.3, centre lines
.delta. 49.0 and .delta. 78.3): .delta. 151.2 (C), 150.2 (CH),
146.0 (C), 128.1 (CH), 115.0 (C), 114.2 (C), 63.6 (CH.sub.2), 59.0
(CH), 40.7 (CH.sub.2), 32.5 (CH.sub.2), 31.6 (CH.sub.2), 30.4
(CH.sub.2), 30.1 (2.times.CH.sub.2), 29.9 (CH.sub.2), 26.8
(CH.sub.2), 23.2 (CH.sub.2), 14.3 (CH.sub.3). ESI-HRMS calcd for
C.sub.18H.sub.32N.sub.5O.sup.+, (M+H).sup.+, 334.2602. found
334.2605.
Example V. Synthesis of
(2R)-2-[({4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-(pyrazi-
n-2-ylsulfanyl)propan-1-ol (V.2)
##STR00034##
[0094] tert-Butyl
(4R)-2,2-dimethyl-4-[(pyrazin-2-ylsulfanyl)methyl]-1,3-oxazolidine-3-carb-
oxylate (V.1). Step 1
[0095] Potassium thioacetate (0.588 g, 5.05 mmol)) and mesylate
from step 1 of the preparation of K.1 (0.520 g, 1.68 mmol) were
stirred together in DMF (5 mL) at rt for 16 h then at 70.degree. C.
for 1 h. Water (5 mL) was added and the mixture extracted with
Et.sub.2O (100 mL). The extract was washed with H.sub.2O (4.times.5
mL), brine (5 mL), dried, the solvent evaporated and the residue
chromatographed on silica gel (gradient of 0-30% EtOAc in hexanes)
to give tert-butyl
(4R)-4-[(acetylsulfanyl)methyl]-2,2-dimethyl-1,3-oxazolidine-3-carboxylat-
e as a colourless oil (0.347 g, 71%)
Step 2
[0096] Sodium methoxide in methanol solution (25%, 0.27 mL, 1.2
mmol) was added to a solution of the thioacetate from step 1 (0.340
g, 1.17 mmol) in MeOH (5 mL)). After 10 mins the solvent was
evaporated and the residue dissolved in DMF (5 mL) then
2-chloropyrazine (0.32 mL, 3.6 mmol) added and the mixture stirred
for 16 h. Water (5 mL) was added then the mixture was extracted
with Et.sub.2O (100 mL). The extract was washed with H.sub.2O
(4.times.5 mL), brine (5 mL), dried and evaporated. The residue was
chromatographed on silica gel (gradient of 0-30% EtOAc in hexanes)
to give V.1 as a colourless oil (0.224 g, 59%). .sup.1H NMR (500
MHz, CDCl.sub.3): .delta. 8.55, 8.48 (2.times. bs, 1H), 8.36, 8.33
(2.times. bs, 1H), 8.22 (bs, 1H), 4.23, 4.14 (2.times. bs, 1H),
4.06-3.93 (m, 2H), 3.76-3.63 (m, 1H), 3.37-3.27 (m, 0.5H),
3.17-3.07 (m, 0.5H), 1.67-1.59 (m, 3H), 1.53-1.45 (m, 12H).
.sup.13C NMR (125.7 MHz, CDCl.sub.3, centre line .delta. 77.0):
.delta. 156.5, 156.1 (C), 152.2, 151.6 (C), 143.7, 143.6
(2.times.CH), 139.8, 139.7 (CH), 94.5, 93.9 (C), 80.5, 80.2 (C),
66.4, 66.3 (CH.sub.2), 56.9, 56.5 (CH), 31.4, 31.0 (CH.sub.2), 28.5
(CH.sub.3), 27.5, 26.9 (CH.sub.3), 24.4, 23.2 (CH.sub.3). ESI-HRMS
calcd for C.sub.15H.sub.23N.sub.3NaO.sub.3S.sup.+ (M+Na).sup.+,
348.1353. found 348.1350.
(2R)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-3-(pyrazin-
-2-ylsulfanyl)propan-1-ol (V.2)
[0097] Compound V.1 (0.220 g, 0.68 mmol) was dissolved in
CH.sub.2Cl.sub.2 (8 mL) and trifluoroacetic acid (2 mL) added.
After 2 h the solvent was evaporated and the residue dissolved in
MeOH (10 mL) and neutralized with Amberlyst A21 resin then passed
through a short column of the same resin and eluted with MeOH. The
fractions containing product were evaporated to a yellow gum that
was dissolved in tert-butanol (4 mL) then aq. formaldehyde solution
(37%, 0.061 mL, 0.81 mmol) and 9-deazaadenine (0.091 g, 0.68 mmol)
were added and the mixture heated at 70.degree. C. for 16 h. Silica
gel was added to absorb all the solvent then the solvent was
evaporated and the residue chromatographed on silica gel (gradient
of 10-15% 7M NH.sub.3/MeOH in CHCl.sub.3) to give V.2 as a
colourless solid (0.055 g, 25%). .sup.1H NMR (500 MHz, CD.sub.3OD):
.delta. 8.35 (d, J=1.5 Hz, 1H), 8.28 (dd, J=2.6, 1.7 Hz, 1H), 8.14
(d, J=2.7 Hz, 1H), 8.12 (s, 1H), 7.43 (s, 1H), 4.05 (d, J=13.9 Hz,
1H), 4.02 (d, J=13.8 Hz, 1H), 3.74 (dd, J=11.3, 4.9 Hz, 1H), 3.64
(dd, J=11.3, 5.5 Hz, 1H), 3.39-3.31 (m, 2H+residual deuterated
solvent), 3.01-2.97 (m, 1H). .sup.13C NMR (125.7 MHz, CD.sub.3OD,
centre line .delta. 49.0): .delta. 158.2 (C), 152.0 (C), 150.8
(CH), 146.5 (C), 145.2 (CH), 144.6 (CH), 140.3 (CH), 129.0 (CH),
115.4 (C), 114.7 (C), 63.7 (CH.sub.2), 58.4 (CH), 41.4 (CH.sub.2),
31.6 (CH.sub.2). ESI-HRMS calcd C.sub.14H.sub.18N.sub.7OS.sup.+
(M+H).sup.+, 332.1289. found 332.1287.
Example W.
(2R)-2-[({4-Amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl}methyl)amino]-
-3-(methylsulfanyl)propan-1-ol (W.1)
##STR00035##
[0099] Prepared according to the literature [Clinch, et
al..sup.18].
Example X (2S)-2-[({4-Amino-5H-pyrrolo[3,
2-d]pyrimidin-7-yl}methyl)amino]-3-(methylsulfanyl)propan-1-ol
(X1)
##STR00036##
[0101] Prepared according to the literature [Clinch, et
al..sup.18].
Example Y. (2R,3S)-2-[({4-Amino-5H-pyrrolo[3,
2-d]pyrimidin-7-yl}methyl)amino]-4-(methylsulfanyl)butane-1, 3-diol
(Y.1)
##STR00037##
[0103] Prepared according to the literature [Clinch, et
al..sup.18].
Example Z. (2S,3R)-2-[({4-Amino-5H-pyrrolo[3,
2-d]pyrimidin-7-yl}methyl)amino]-4-(methylsulfanyl)butane-1, 3-diol
(Z.1)
##STR00038##
[0105] Prepared according to the literature [Clinch, et
al..sup.18].
Example AA. Synthesis of
7-[(octylamino)methyl]-5H-pyrrolo[3,2-d]pyrimidin-4-amine
(AA.1)
##STR00039##
[0106] 7-[(Octylamino)methyl]-5H-pyrrolo[3, 2-d]pyrimidin-4-amine
(AA.1
[0107] Octan-1-amine (0.100 g, 0.77 mmol), aq. formaldehyde
solution (37%, 0.076 mL, 1.01 mmol) and 9-deazaadenine (0.105 g,
0.78 mmol) were heated in tert-butanol (3 mL) at 70.degree. C. for
16 h. Silica gel was added to absorb all the solvent then the
solvent was evaporated and the residue chromatographed on silica
gel (gradient of 5-15% 7 M NH.sub.3/MeOH in CHCl.sub.3) to give
AA.1 as a colourless solid (0.132 g, 62%). .sup.1H NMR (500 MHz,
CD.sub.3OD): .delta. 8.16 (s, 1H), 7.46 (s, 1H), 3.91 (s, 2H), 2.61
(t, J=7.5 Hz, 2H), 1.52 (pent, J=7.4 Hz, 2H), 1.34-1.21 (m, 10H),
0.88 (t, J=7.1 Hz, 3H). .sup.13C NMR (125.7 MHz, CD.sub.3OD centre
line .delta. 49.0): .delta. 152.1 (C), 150.8 (CH), 146.6 (C), 129.0
(CH), 115.4 (C), 114.5 (C), 49.9 (CH.sub.2), 43.7 (CH.sub.2), 33.0
(CH.sub.2), 30.5 (CH.sub.2), 30.4 (CH.sub.2), 30.3 (CH.sub.2), 28.4
(CH.sub.2), 23.7 (CH.sub.2), 14.4 (CH.sub.3). ESI-HRMS calcd for
C.sub.15H.sub.26N.sub.5.sup.+, (M+H).sup.+, 276.2183. found
276.2181.
RESULTS AND DISCUSSION
[0108] Commonly used antibiotics in H. pylori infections include
amoxicillin, metronidazole and tetracycline. The anti-H pylori
effects of selected compounds were compared to those antibiotics in
common use.
[0109] In most bacteria, MTANs are expressed and catalyze the
hydrolysis of the N-ribosidic bonds of 5'-methylthioadenosine and
S-adenosylhomocysteine. The two reactions are involved in bacterial
quorum sensing, sulfur recycling via S-adenosylmethionine and
polyamine synthesis.sup.13; however, most bacterial MTANs are not
essential for bacterial proliferation as judged by planktonic
growth conditions.
[0110] Bacterial genome analysis predicts the HpMTAN-mediated
pathway for menaquinone biosynthesis to be rare, but also to be
present in Campylobacter species.sup.4. Campylobacter jejuni is the
world's leading cause of bacterial gastroenteritis.sup.11.
[0111] Drug resistance has developed quickly in H. pylori, and
currently, approximately 30% of H. pylori infection are resistant
to single-agent first line drugs.sup.12. As a result, the current
approach commonly uses triple-agent therapy for H. pylori
infections and includes two antibiotics with different mechanisms
of action. Even with triple-agent therapy, more than 20% of H.
pylori infections are not readily eradicated.sup.2. Resistance in
the H. pylori population is no doubt partially due to exposing H.
pylori to broad spectrum antibiotics during the treatment of other
bacterial infections. In addition, current eradication of H. pylori
requires antibiotics for two weeks or longer and there is an
increase in the development of resistance if treatment is
interrupted. Table 1 summarizes the dissociation constants versus
H. pylori MTAN and the MIC90 values against H. pylori for specific
compounds of the invention. Drug combinations using these compounds
may also address current issues of antibiotic resistance.
TABLE-US-00001 TABLE 1 Acyclic Amine Inhibitors of Helicobacter
pylori MTAN and their MIC90 values against H. pylori. ##STR00040##
##STR00041## 0.10 .+-. 0.01 8 ##STR00042## 0.10 .+-. 0.01 8
##STR00043## 0.030 .+-. 0.003 8 ##STR00044## 0.11 .+-. 0.01 16
##STR00045## 0.9 .+-. 0.2 16 ##STR00046## 1.2 .+-. 0.4 40
##STR00047## 0.12 .+-. 0.01 80 ##STR00048## 0.7 .+-. 0.1 0.21 .+-.
0.02 >80 ##STR00049## 0.8 .+-. 0.1 >80 ##STR00050## 0.9 .+-.
0.1 >80 ##STR00051## 5 .+-. 2 >80 ##STR00052## 13 .+-. 2
>80 ##STR00053## 13 .+-. 2 >80 ##STR00054## >5 >80
##STR00055## >10 >80 ##STR00056## >50 >80 ##STR00057##
0.05 .+-. 0.01 80 ##STR00058## >5 >80 ##STR00059## >5
>80 ##STR00060## >5 >80 ##STR00061## >5 >80
##STR00062## >5 >80
TABLE-US-00002 TABLE 2 Other Acyclic Amine Inhibitors of
Helicobacter pylori MTAN and their MIC90 values against H. pylori.
##STR00063## ##STR00064## 0.2 .+-. 0.04 >80 ##STR00065## >5
>80
Example 2
In Vitro Activity of Compound T.2 (Hexyl-SerMe-Immucillin A)
[0112] These following experiments were conducted at UNT Health
Science Center (Study lead: William J Weiss, Director of
Pre-Clinical Services), Fort Worth, Tex., USA.
[0113] (a) Sample Plate Preparation:
[0114] Aliquots of a 2-fold serial dilution of
Hexyl-SerMe-Immucillin A (from 62.5 .mu.g/mL-6.25 .mu.g/mL in DMSO,
0.02 mL) were mixed into separate 2 mL molten MHIIb agar, and
poured into separate wells on each well of a 12-well plate, allowed
to set for 20-30 minutes and dry in a biosafety cabinet for 10
minutes prior to Inoculation.
[0115] (b) Inoculation:
[0116] Frozen inoculate stock of H. pylori strains UNT020-1 (Sydney
Strain SS1) and UNT189-1 (ATCC43504) was separately streaked onto
Columbia+5% sheep blood agar and microaerophilically incubated for
72 h at 37.degree. C. The plate cultures of the H. pylori strains
were then harvested in 0.9% sterile saline solution and the OD at
530 nm of the suspension was determined. The OD of each suspension
was adjusted to 1.5-2.0 by dilution into 0.9% sterile saline. The
UNT020-1 suspension was further diluted 2:5, but the UNT189-1
suspension was not further diluted, and these were used to
inoculate the agar plates. Samples of the suspensions (3 .mu.L)
were spotted onto the MHIIb agar wells in the 12-well plate. The
CFU/mL of each inoculum was confirmed by generating a 10-fold
serial dilution of each inoculum in 0.9% sterile saline and
spotting 8 .mu.L of each dilution onto Columbia+5% sheep blood agar
plates. After allowing the spots to dry for 10 min, the plates were
placed into a microaerophilic chamber and incubated at 37.degree.
C. for 72 h.
[0117] Results:
[0118] The Minimum Inhibitory Concentrations (MICs) for the
Hexyl-SerMe-immucillin A was determined against H. pylori isolates
UNT020-1 (Sydney Strain SS1) and UNT189-1 (ATCC43504).
Hexyl-SerMe-Immucillin A exhibited excellent activity against the
two H. pylori strains with an MIC of 1.9 ng/mL in each case.
The Concentration of Compound T.2 (Hexyl-SerMe-Immucillin A) in the
Gastric Mucin of Mouse Following a Single Oral Dose at 10 mg/kg
[0119] These following experiments were conducted at UNT Health
Science Center (Study lead: William J Weiss, Director of
Pre-Clinical Services), Fort Worth, Tex., USA.
[0120] (a) Oral Dosing of Mice with 10 mg/kg Hexyl-SerMe-Immucillin
A:
[0121] Female C57/BL6 mice ranging from 5-6 weeks in age and 18-22
grams in weight (Harlan Laboratories) were dosed by oral gavage
with of Hexyl-SerMe-Immucillin A (0.4 mL of a 0.5 mg/mL solution in
water for injection).
[0122] (b) Mucin Sampling:
[0123] Mice (3 mice/time point) were euthanized via CO.sub.2
inhalation at 0.25, 0.5, 1, 2, 4, 8, 12 and 24 h, then the mucin
layer was scraped from the longitudinally dissected stomach using a
glass slide, collected and weighed.
[0124] (c) Preparation of Gastric Mucin Homogenates:
[0125] Approximately 30 mg of mouse mucin was weighted into a 2 mL
homogenizer tube (prefilled with 3.0 mm i.d. zirconium beads) and
mixed with 0.2 mL PBS buffer. This was homogenized for 3 min at
4,000 rpm in a Beadbug D1030 (Benchmark Scientific)
homogenizer.
[0126] (d) Extraction of Gastric Mucin Homogenates:
[0127] Samples of mucin homogenate (50 .mu.L) and of internal
standard (see below) in water (10 .mu.L) were treated with
trichloroacetic acid (30% w/v, 30 .mu.L), diluted with water (160
.mu.L), vortexed for 10 sec and sonicated for 10 min, then
centrifuged at 14,500 rpm for 5 min. A sample of the supernatant
(160 .mu.L) was transferred into autosampler vials. The internal
standard was Butylthio-DADMe-Immucillin-H at 5.0 and 20.0
.mu.g/mL.
[0128] (f) HPLC-MS-MS Analysis:
[0129] Aliquots of supernatant (10 .mu.L) were injected for
analysis on Surveyor HPLC system (Thermo) fitted with an ACE 3 C18
Column (50.times.3 mm, 3 .mu.m particles), with gradient elution at
30.degree. C. with 400 .mu.L/min mobile phase comprised of mixtures
of 0.1% trifluoroacetic acid in water (A) and methanol (B) as
follows: 0-5 min, A:B 9:1; 5-6.1 min, A:B 1:9; 6.1-10 min A:B 9:1.
Hexyl-SerMe-Immucillin A eluted at 4.41.+-.0.003 min. Detection was
ESI MS/MS using a LCQ Deca (Thermo) in selected-reaction monitoring
(SRM) positive mode using the MS/MS event m/z 292.2.fwdarw.147.2
and ion spray voltage of 4.5 kV.
[0130] (g) Results:
[0131] The results for Hexyl-SerMe-Immucillin A in gastric mucin
following administration of 10 mg/kg by oral gavage in mice are
shown in the Table and FIGURE below. A high and prolonged level of
Hexyl-SerMe-Immucillin A was detected in the gastric mucin, with
the C.sub.max value was 65.5 .mu.g/g, total exposure
[AUC.sub.(0-inf)] was 78.6 .mu.g-h/g and the terminal half-life was
2.72 h.
TABLE-US-00003 TABLE 3 The concentration of Hexyl-SerMe-Immucillin
A in mouse gastric mucin versus time following a single dose of 10
mg/kg PO. Time Hexyl-SerMe-Immucillin A (.mu.g/g) (h) Animal-1
Animal-2 Animal-3 Mean SD SEM 0.25 60.42 64.36 45.91 56.89 9.71
4.34 0.50 46.24 90.18 60.18 65.53 22.46 10.04 1.00 20.39 12.20
12.04 14.88 4.77 2.13 2.00 2.02 1.08 16.24 6.44 8.49 3.80 4.00
0.67* 3.74 0.91 1.77 1.71 0.76 8.00 0.65* 1.62 0.53* 0.93 0.60 0.27
12.0 0.96 0.95 0.33* 0.74 0.36 0.16 24.0 0.54* 0.39* 0.30* 0.41
0.12 0.06 *below LLOQ
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